This invention relates to α-conotoxin peptides, and in particular to cyclised α-conotoxin peptides useful in the therapeutic treatment of humans. The invention especially relates to oral and enteral preparations comprising these peptides, the use of these peptides in the manufacture of pharmaceutical preparations, and the use of these pharmaceutical preparations in the prophylaxis or treatment of conditions or diseases in humans.
The marine snails of the genus Conus (cone snails) use a sophisticated biochemical strategy to capture their prey. As predators of either fish, worms or other molluscs, the cone snails inject their prey with venom containing a cocktail of small bioactive peptides. These toxin molecules, which are referred to as conotoxins, interfere with neurotransmission by targeting a variety of ion-channels or receptors. They typically contain 12-30 amino acids arranged in linear sequence. The venom from any single Conus species may contain more than 100 different peptides. The conotoxins are divided into classes on the basis of their physiological targets. For example, the α-conotoxin and Ψ-conotoxins target nicotinic ACh receptors, causing ganglionic and neuromuscular blockade, while the ω-conotoxin class of peptides target voltage-sensitive Ca2+-channels, inhibiting neurotransmitter release.
Most conotoxin peptides contain either four (4) or six (6) cysteine residues which are bonded in pairs to form either two (2) or three (3) disulfide bonds respectively, although there are some examples having two cysteine residues bonded to form a single disulfide bond (i.e., conoptessins), as well as some having greater than three disulfide bonds, and others that contain no cysteine residues or disulfide bonds. The peptides of some of the “activity” classes described above share a structural motif, possessing the same number of cysteine residues and the same disulfide bond connectivity. For this reason a new “superfamily” classification system has been developed. For example, the ω-conotoxins and members of the δ and μ-conotoxin classes have six cysteine residues which are bonded in pairs to form three disulfide bonds between cysteine residues I and IV, II and V, and III and VI, where the six Roman numerals represent the six cysteine residues numbering from the N-terminus. Conotoxin peptides having this structural motif belong to the O-superfamily and M-superfamily of conotoxins. Similarly, ρ-conotoxins and most α-conotoxins have four cysteine residues bonded in pairs to form two disulfide bonds between cysteine residues I and III, and II and IV. These conotoxin peptides belong to the “A-superfamily” of conotoxins. The present invention relates to α-conotoxins in the A-superfamily, i.e. α-conotoxin peptides having two disulfide bonds formed between cysteine residues I and III, and II and IV. As indicated above, conotoxin peptides bind to a range of different ion channel receptors in mammals and accordingly they have several potential therapeutic applications, including pain relief in humans. However, in general peptides have several difficulties associated with their use as drugs, including generally poor bioavailability; susceptibility to cleavage by proteases, and unwanted side effects.
One ω-conotoxin, MVIIA (also known as SNX-111, Ziconitide and Prialt), recently received approval by the United States Food and Drug Administration for the treatment of intractable pain associated with cancer, AIDS and neuropathies. The route of administration is currently restricted to intrathecal infusion into the spinal cord because of some of the abovementioned difficulties, and because the receptors targetted by this drug are located within the CNS.
Another ω-conotoxin which has commenced clinical trials is CVID (AM336—Zenyth Pharmaceuticals) which is reported to have improved selectivity for N-type calcium channels over P/Q-type channels relative to Ziconitide. However, even with improved selectivity for particular receptors/channels, conotoxins of the ω class have still been associated with undesirable side effects in some patients. Two other conotoxin peptides (CGX-1160 and CGX-1007 isolated from Conus geographus) are also undergoing clinical trials, however administration of these peptides is also restricted to the intrathecal route. Another peptide being investigated for pain is a conotoxin of the χ-class (Xen 2174-Xenome Ltd). Again, administration of this peptide is limited to the intrathecal route.
The main disadvantages of intrathecal or spinal administration of drugs are that administration must be carried out by a doctor or nurse, and for long term treatment a mechanical delivery system and spinal catheter are preferably inserted into the spine of a patient. Hence, such treatment is usually reserved for the terminally ill and/or hospital-bound patients.
The α-conotoxins are a sub-family of conotoxins that typically range in size from 12 to 16 amino acids, and usually have an amidated C-terminus. The α-conotoxins are known to inhibit nicotinic acetylcholine receptors. The α-conotoxins interact with both muscle and neuronal nicotinic acetylcholine receptors (nAChRs) which have been implicated in a range of disorders including Alzheimer's disease, schizophrenia, depression and small cell lung carcinoma, as well as playing a role in analgesia and addiction. α-conotoxin peptides and their potential uses are widely described in the literature (see Lloyd and Williams, 2000, J Pharmacol Exp Ther 292 (2) 461).
The number of residues between the cysteine residues is used to distinguish different classes of α-conotoxins. Based on the number of residues between the second and third cysteine residues (loop 1) and the third and fourth residues (loop 2) they are divided into α3/5, α4/3, α4/4, α4/6 and α4/7 structural subfamilies. Two examples of α-conotoxin peptides having 4/7 loop arrangement are MII and Vc1.1 (also known as ACV1—Metabolic Pharmaceuticals Limited). One of the smallest α-conotoxin peptides is IM1 which has a 4/3 loop arrangement.
Several α-conotoxin peptides have been studied to ascertain their selectivity for the various subtypes of nicotinic acetylcholine receptors, and in particular their selectivity for peripheral nAChR subtypes over central subtypes. nAChRs are expressed at low levels throughout the CNS and PNS, but various subtypes have different distributions. The mammalian nAChRs are composed of combinations of subunits. Seven of these subunit types are the major components involved in ligand binding (α2, α3, α4, α6, α7, α9 and α10) while 4 subunit types (α5, β2, β3 and β4) are considered to be structural, imparting functional and pharmacological properties to the receptors. The different subtypes combine in a variety of ways (generally as heterologous pentamers) to form receptors having particular pharmacological and electrophysiological properties. The α3 subunit is considered to be a peripheral subunit (due to its presence in the PNS) while the α7 subunit is considered to be a subunit prevalent in the CNS.
The α-conotoxin Vc1.1 was first discovered using a PCR screen of cDNAs from the venom ducts of Conus victoriae (Sandall et al. Biochemistry, 2003, 42, 6904). The cysteine spacing within the sequence of Vc1.1 indicates that it is a member of the 4/7 subclass of α-conotoxins, which includes the extensively studied conotoxins MII, EpI and PnIB. The three dimensional structure of Vc1.1 comprises a small α-helix spanning residues P6 to D11 and is braced by the I-II, III-IV disulfide connectivity seen in other α-conotoxins (Clark et al., J. Biol. Chem. 2006, 281, 23254). In addition to an amidated C-terminus, which is common to most α-conotoxins, it is also possible to postranslationally modify residues Pro6 and Glu14 in linear Vc1.1 to hydroxyproline and γ-carboxyglutamate respectively. This post translationally modified analogue of Vc1.1 is implicated in nerve regeneration but not pain (WO 02/079236).
Linear Vc1.1, an antagonist of neuronal nAChRs in bovine chromaffin cells, has been shown to alleviate neuropathic pain in three rat models of human neuropathic pain and to accelerate the functional recovery of injured neurons (Satkunanathan et al., 2005, Brain Research 1059 (2) 149-158). In addition it has been reported by Livett et al that ACV-1 is effective at alleviating neuropathic pain in an animal model of diabetic neuropathy. In particular, in the streptozotocin-induced diabetic rat model of peripheral neuropath an anti allodynic effect of ACV-1 was observed at doses of 30 and 300 ug/kg within 1 hr of dosing (IDrugs, 15th World Congress on Animal, Plant and Microbial Toxins, 2006 9, 679-681). As an analgesic, Vc1.1 has been reported be more active than Ziconotide (Sandall et al., 2003, Biochemistry, 42, 6904-6911). More recently, Vc1.1 was shown to antagonize the nicotine-induced increase in axonal excitability of unmyelinated C-fiber axons in isolated segments of peripheral human nerves (Lang et al., 2005, Neuroreport 16, 479-483). As mentioned above neuronal nAChRs are pentameric ligand-gated ion channels composed of combinations of α (α2 to α10) and β (β2 to β4) subunits that are found throughout both the central and peripheral nervous systems. Electrophysiological and immunohistochemical data indicate the functional expression of nAChRs composed of α3, α5 and β4 but not α4, β2 or α7 subunits in axons of unmyelinated C fibers (Lang et al., 2005, Neuroreport 16, 479-483; and Lang et al., 2003, Neurophysiol 90, 3295-3303). Blockade of nAChRs on unmyelinated peripheral nerve fibers may have an analgesic effect on unmyelinated sympathetic and/or sensory axons. Interestingly, synthetic post translationally modified Vc1.1 (ptmVc1.1) was reported to not inhibit the neuronal-type nicotinic response in chromaffin cells and was inactive in two rat neuropathic pain assays. Linear Vc1.1 or ACV1 has commenced phase 2 human clinical trials. Nicotinic agonists have been previously reported to possess analgesic activity. Examples of such nicotinic agonists are epibatadine and ABT-594. It is postulated that these agents act by desensitising the nicotinic receptor, resulting in a reduction of ion flux through the receptor. Under these conditions the agonists are effectively acting as antagonists of the nAChRs and for this reason antagonists of nAChRs have been sought as potential analgesic compounds. Conotoxin peptide Vc1.1 is said to be such an antagonist.
Interestingly, the IC50 values for Vc1.1 at the α3, β4 subtype of the nAchR is in the micromolar range (4.2 μM; Vinvcler et al., 2006, PNAS, 103, 17881). The analgesic effect of Vc1.1 appears to occur in nanomolar concentrations in vivo.
It has recently been reported that Vc1.1 may produce analgesia through the modulation of α9, α10 nAchRs (Vincler et al., supra). The affinity of Vc1.1 for the α9, α10 nAchR is one hundred fold higher than for the α3, β4 nAchR and falls in the nanomolar range (22.9 nM). Therefore, it appears that one physiological target for Vc1.1 may be the α9, α10 nAchR which is known to have widespread distribution, and located in dorsal root ganglion, neurones, the pituitary, lymphocytes, skin keratinocytes and sperm. However, it is conceivable that there may be other targets yet to be discovered that are responsible for the pain-relieving activity of Vc1.1.
Unlike the previous conotoxin peptides which have been investigated for the treatment of pain and other conditions, Vc1.1 is said to have the advantage that it can be administered subcutaneously or intramuscularly, rather than intrathecally. This is said to provide a significant advantage for Vc1.1 over previous conotoxin peptides, including Ziconitide. However, conotoxin peptide Vc1.1 is said to lack oral bioavailability. According to a document published on Metabolic Pharmaceuticals' website dated January 2006 “ACV1—A novel therapeutic for neuropathic pain, Technical Summary of Preclinical Data” ACV1 is not orally available and current development is as a subcutaneous injectable treatment. Vc1.1 is also reported to be effective in an animal model of inflammatory pain and to accelerate the recovery of injured nerves and tissues.
WO 2007/014432, in the name of Metabolic Pharmaceuticals Limited, describes a method for improving the oral delivery of a peptide drug by linking the C terminal sequence of human growth hormone and analogues of same to the C terminal of the peptide drug. Addition of the C terminal sequence of human growth hormone to a peptide drug allegedly confers oral bioavailability properties to the peptide drug. The patent application describes an orally available peptide that appears to be active in an animal model of neuropathic pain. The peptide comprises Vc1.1 with the amino acid sequence Tyr-Leu-Arg-Ile-Val linked to the C terminus of Vc1.1.
Accordingly, there is still a need for effective method of treating patients with α-conotoxin peptides via oral or enteral routes, particularly in relation to the production of analgesia, the treatment or prevention of neuropathic pain and in the acceleration of recovery from nerve injury.